By William R. Skach
Cystic Fibrosis: tools and Protocols consolidates state-of-the-art in vitro, mobile, and full animal laboratory protocols into an integral source. From electrophysiology and phone biology, to animal types and gene remedy, this entire set of tools presents the step by step directions wanted for investigators to include new methods into their examine courses. particular protocols describe new options for prognosis, in vitro equipment for the expression and sensible research of CFTR, novel biochemical and mobile structures to figure out how mutations subvert CFTR functionality, and in vivo protocols to envision how CFTR disorder produce multisystem pathology in either human and animal types.
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22. Welsh, M. J. and Smith, A. E. (1993) Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73, 1251–1254. 23. Choi, J. , Lee, M. , Thomas, P. , and Muallem. S. (2001) Aberrant CFTR-dependent HCO-3 transport in mutations associated with cystic fibrosis. Nature 410, 94–97. 24. Tebbutt, S. , Wardle, C. , Hill, D. , and Harris, A. (1995) Molecular analysis of the ovine cystic fibrosis transmembrane conductance regulator gene. Proc. Natl. Acad. Sci. USA 92, 2293–2297.
However, variations of this simplest pattern can be observed if a portion of one or the other strand assumes a second stable configuration. Regardless, if a mutation exists on one chromosome, it almost always alters the migration pattern of one or both strands, as well as forming heteroduplexes with the unmutated opposite strands. The method depends simply on being able to detect this different migration. We use gel conditions that had previously been optimized for CFTR and that should be capable of detecting >95% of CFTR mutations (45,46).
F. 6%C). g. Tris-Borate–EDTA (TBE) buffer, 5× concentration (Sigma). h. 1 L of 1× TBE buffer was used for electrophoresis. 11. Electrophoresis power supply, EPS 1001 (Amersham Pharmacia Biotech, Piscataway, NJ) was used for electrophoresis. 12. Autoradiography was done on Kodak Scientific Imaging Film X-OMAT AR (35 × 43 cm) (Eastman Kodak Company, Rochester, NY). 13. Mutations were made with Stratagene’s Quick-change site-directed mutagenesis kit (La Jolla, CA) and verified with restriction enzymes (Life Technologies, Grand Island, NY) or sequencing.
